Objective Prostate-specific antigen (PSA) is the best established biomarker of semen exposure. PSA in vaginal fluid returns to pre-exposure concentrations within 24–48 h, but the speed of decay during the first 10 h is unknown. We sought to determine how fast PSA concentrations decline during the first 10 h after exposure to semen. Study design Women in the 50 enrolled couples were intravaginally inoculated with 10, 20, 100 and 200 μl of their partner's semen and then collected vaginal swabs immediately after, 30 min, 4 h and 10 h after exposure. Forty-seven sets of samples were tested for PSA. Mixed linear models for repeated measures examined the association between log-transformed PSA values and sampling time and semen exposure volume. Sensitivity analyses excluded data from nonabstainers. Fixed-effect estimates from the statistical models were graphed. Results PSA values were highest at 200 μl inoculation volumes and at earlier post-exposure time points, then decline steadily. The lowest inoculation volume (10 μl) corresponded to the smallest concentration of PSA throughout the post-inoculation time points. Average PSA levels return to clinically non-detectable levels within 10 h only at the lowest semen exposures. The PSA decay curve assumes a very similar profile across all time points and semen amounts. Conclusions The PSA decay curve is similar for varying semen exposure volumes, with average PSA concentrations remaining above clinical thresholds 10 h after exposure at all except the very smallest semen exposure levels. PSA is an objective marker of recent exposure to semen, permitting such detection with high accuracy. Implications This study clarifies how PSA values vary at different semen exposure levels and time points during the first 10 h post-exposure. Future contraceptive studies that use PSA as a semen biomarker will be better informed about PSA concentrations at different sampling times and exposure amounts.